Hemostasis is the normal physiological process in which bleeding from an injured blood vessel is arrested. It is a dynamic and complex process in which proteolytic enzymes such as thrombin play a key role. Blood coagulation may occur through either of two cascades of zymogen activations, the extrinsic and intrinsic pathways of the coagulation cascade. Factor VIIa in the extrinsic pathway, and Factor IXa in the intrinsic pathway are important determinants of the activation of factor X to factor Xa, which itself catalyzes the activation of prothrombin to thrombin. The last protease in each pathway is thrombin, which acts to hydrolyze four small peptides (two FpA and two FpB) from each molecule of fibrinogen, thus deprotecting its polymerization sites. Once formed, the linear fibrin polymers may be cross-linked by factor XIIIa, which is itself activated by thrombin. In addition, thrombin is a potent activator of platelets, upon which it acts at specific receptors. Thrombin activation of platelets leads to aggregation of the cells and secretion of additional factors that further accelerate the creation of a hemostatic plug. Thrombin also potentiates its own production by the activation of factors V and VIII (see Hemker and Beguin in: Jolles, et. al., "Biology and Pathology of Platelet Vessel Wall Interactions," pp. 219-26 (1986), Crawford and Scrutton in: Bloom and Thomas, "Haemostasis and Thrombosis," pp. 47-77, (1987), Bevers, et. al., Eur. J. Biochem. 1982, 122, 429-36, Mann, Trends Biochem. Sci. 1987, 12, 229-33).
Thrombosis may be regarded as the pathological condition wherein improper activity of the hemostatic mechanism results in intravascular thrombus formation. Etiological factors such as the presence of atherosclerotic plaque, phlebitis and septicemia may cause thrombosis, leading to impaired blood flow to the effected tissues and possible serious pathological consequences. Thrombosis may be reduced by inhibition of the normal process of blood coagulation by anticoagulants. Anticoagulants act by reducing the amount of thrombin which is generated, or by inhibiting with the proteolytic actions of thrombin.
Currently, two of the most effective classes of drugs in clinical use as anticoagulants are the heparins and the vitamin K antagonists. The heparins are ill-defined mixtures of sulfated polysaccharides that bind to, and thus potentiate the action of antithrombin III. Antithrombin III is a naturally occurring inhibitor of the activated clotting factors Ixa, Xa, XIa, thrombin and probably XIIa (see Jaques, Pharmacol. Rev. 1980, 31, pp. 99-166). The vitamin K antagonists, of which warfarin is the most well-known example, act indirectly by inhibiting the post-ribosomal carboxylations of the vitamin K dependent coagulation factors II, VII, IX and X (see Hirsch, Semin. Thromb. Hemostasis 1986, 12, 1-11). While effective therapies for the treatment of thrombosis, heparins and vitamin K antagonists have the unfortunate side effects of bleeding and marked interpatient variability, resulting in a small and unpredictable therapeutic safety margin. The use of direct acting thrombin inhibitors is expected to alleviate these problems.
Anticoagulants are also necessary in the processing of blood for therapeutic or diagnostic purposes or for the production of blood products or fragments, since contact of blood with the surfaces commonly used for blood collection and storage causes activation of coagulation leading to thrombin formation and clot formation.
The coagulation proteases thrombin, factor Xa, factor VIIa, and factor IXa are serine proteases having trypsin-like specificity for the cleavage of sequence-specific Arg-Xxx peptide bonds. As with other serine proteases, the cleavage event begins with an attack of the active site serine on the scissile bond of the substrate, resulting in the formation of a tetrahedral intermediate. This is followed by collapse of the tetrahedral intermediate to form an acyl enzyme and release of the amino terminus of the cleaved sequence. Hydrolysis of the acyl enzyme then releases the carboxy terminus.
A number of naturally occurring thrombin inhibitors have been reported. These include nazumamide A from Theonella sp. (see Fusetani, et. al., Tetrahedron Lett. 1991, 32, 7073-4), cyclotheonamide A from Theonella sp. (see Fusetani, et. al., J. Am. Chem. Soc. 1990, 112, 7053-4), amblyommin from Amblyomma hebraeum (see Bonin, et. al., EP 345614), hirudin from Hirudo medicinalis, recombinant versions of hirudin and hirudin fragments (see Rigbl and Jackson, EP 352903, Koerwer, WO 9109946, Meyer, et. al., WO 9108233, Dawson, et. al., WO 9109125, Maraganore, et. al., WO 9102750 and Maraganore, EP 33356).
Synthetic thrombin inhibitors have also been disclosed. Arylsulfonylarginine amides such as (2R,4R)-4-methyl-1-[N.sup.2 -{(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl) sulfonyl}-L-arginyl]-2-piperidinecarboxylate have been shown to be effective inhibitors of thrombin (see Okamoto, et. al. Thromb Res. 1976, 8, 77-82, Ohshiro, et. al., Blood Vessel 1983, 14, 216-8), as have compounds containing constrained arginine mimics such as (2-naphthylsulfonylglycyl)-4-amidino-phenylalanyl piperidide (see Stuerzebecher, et. al., Thromb. Res. 1983, 29, 635-42), 1-[2-[5-(dimethylamino)naphth-1-ylsulfonamido]-3-(2-iminohexahydropyrimidi n-5-yl)propanoyl]-4-methylpiperidine dihydrochloride (see Ishikawa, JP 88227572 and Ishikawa and Inamura, JP 88227573), N-(trans-4-amino-methylcyclohexylcarbonyl)-4-O-(2-picolyl)-L-tyrosine 4-acetanilide dihydrochloride (see Okamoto, et. al., EP 217286) and 4-[(aminoiminomethyl)amino]benzoic acid esters (see Fuji, et. al., DE 3005580, Matsuoka, et. al., Jpn. J. Pharmacol. 1989, 51, 455-63, and Takeshita, et. al., EP 435235).
Inhibitor design has benefitted from the knowledge of the mechanism of action and of the peptide sequences which are thought to bind in the catalytic site of thrombin, e.g., -Gly-Val-Arg-Gly- of fibrinogen (see Blomb ack, et. al., J. Biol. Chem., 1972, 247, 1496-512), Ile-Pro-Arg-Ser- of prothrombin (see Magnussen, et. al., in: Reich, et. al., "Proteases and Biological Control," pp. 123-149 (1975)) and -Val-Pro-Arg-Gly- of factor XIII (see Takagi and Doolittle, Biochemistry 1974, 13, 750-6 and Nakamura, et. al., Biochem. Biophys. Res. Commun. 1974, 58, 250-256). This class of mechanism-based inhibitors are exemplified by the tripeptide aldehyde D-Phe-Pro-N-Me-Arg-H (see Bajusz, et. al., J. Med. Chem. 1990, 33, 1729-35), the chloromethyl ketone Ac-D-Phe-Pro-ArgCH.sub.2 Cl (see Kettner and Shaw, Thromb. Res. 1979, 14, 969-73) and the trifluoromethyl ketone D-Phe-Pro-ArgCF.sub.3 (see Kolb, et. al., US 697987).
Kettner and Shenvi (EP 293881, published Jun. 12, 1988), disclose peptide boronic acid inhibitors of trypsin-like proteases of formula (1) EQU R.sup.1 -[(A.sup.3).sub.q (A.sup.2).sub.p (A.sup.1).sub.o ].sub.n --NH--CHR.sup.2 --BY.sup.1 Y.sup.2 ( 1)
wherein Y.sup.1 and Y.sup.2, independently, are hydroxyl or fluoro or, taken together, form a moiety derived from a dihydroxy compound having at least two hydroxy groups separated by at least two connecting atoms in a chain or ring, said chain or ring comprising 1 to about 20 carbon atoms and, optionally, a heteroatom which can be N, S, or O; R.sup.2 is a substituted alkyl selected from the group consisting of --(CH.sub.2).sub.z --X, --(CH(CH.sub.3)--(CH.sub.2).sub.2 --X, --CH.sub.2 --CH--(CH.sub.3)--CH.sub.2 --X, --(CH.sub.2).sub.2 --CH(CH.sub.3)--X and --(CH.sub.2).sub.2 --CH(CH.sub.3).sub.2 --X, where X is --NH.sub.2, --NH--C(NH)--NH.sub.2 or --S--C(NH)--NH.sub.2, and z is 3 to 5; n, o, p and q are, independently, either 0 or 1; A.sup.1, A.sup.2 and A.sup.3 are, independently, amino acids of L- or D-configuration selected from the group consisting of Ala, Arg, Ash, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; and R.sup.1 is a peptide comprised of 1 to about 20 amino acids, an acyl or a sulfonyl group comprised of 1 to about 20 carbon atoms, H, or an N-terminal protecting group. In this disclosure, Kettner and Shenvi demonstrated that the pinanediol esters of boropeptides are pharmacogolically equivalent to the corresponding boronic acids.
Metternich (EP 0471651 A2) discloses borolysine thrombin inhibitors of formula (2) EQU W--Y--NR.sup.4 --CHR.sup.5 --BQ.sup.1 Q.sup.2 ( 2)
wherein W is an N-protecting group; Y is a sequence of n amino acids such that the n+1 amino acid peptide Y-Lys or Y-Arg has an affinity for the active site of a trypsin-like protease; where n is an integer of from 1 to 10 and in which at least one amino acid is an unnatural amino acid having a hydrophobic side chain; Q.sup.1 and Q.sup.2 are the same or different and are selected from --OH, --COR.sub.1, --CONR.sub.1 R.sub.2, --NR.sub.1 R.sub.2 or --OR.sub.3 of Q.sup.1 and Q.sup.2 taken together form a diol residue; R.sub.1, R.sub.2 and R.sub.3 which may be the same or different, are C.sub.1-10 alkyl, C.sub.6-10 aryl, C.sub.6-10 aralkyl, or phenyl substituted by up to three groups selected from C.sub.1-4 alkyl, halogen and C.sub.1-4 alkoxy; R.sub.4 is hydrogen or C.sub.1-10 alkyl; R.sub.5 is a group --A--X; wherein A is --(CH.sub.2).sub.z -- in which z is 2, 3, 4 or 5; --CH(CH.sub.3)--(CH.sub.2).sub.2 --; --CH.sub.2 --CH(CH.sub.3)--CH.sub.2 --; --(CH.sub.2).sub.2 --CH(CH.sub.3)--; --(CH.sub.2).sub.2 --C(CH.sub.3) .sub.2 --; CH(CH.sub.3)--(CH.sub.2).sub.3 --; --CH.sub.2 --CH(CH.sub.3)--(CH.sub.2).sub.2 --; --CH.sub.2 --CH.sub.2 --CH (CH.sub.3)--CH.sub.2 --; --(CH.sub.2).sub.3 --CH(CH.sub.3)--; --(CH.sub.2).sub.3 --C(CH.sub.3).sub.2 : C.sub.6-10 aryl C.sub.6-10 aralkyl and X is --NH.sub.2, --NH--C(NH)--NH.sub.2, --S--C(NH)--NH.sub.2, N.sub.3, --C.sub.1-4 alkoxy, C.sub.1-4 alkylthio or Si(CH.sub.3).sub.3 or R.sub.4 and R.sub.5 taken together form a trimethylene group and the asymmetric carbon atom may have the D- or L-configuration or represent any mixture of these.
Surprising for their lack of a basic residue at P.sub.1 are tripeptide thrombin inhibitors comprised of 1-aminoboronic and 1-aminophosphonic acid analogs of 3-methoxy-propylglycine (see Claeson, et. al., U.S. Pat. No. 07-245428) and pentylglycine (see Cheng, et. al., "Symposium on Thrombosis and Hemostasis," 1991, Amsterdam, Abstract 2150).
In addition to thrombin inhibition, boropeptides have been disclosed with utility as a treatment for tumors, viral infections and arthritis (U.S. Pat. No. 4963655A and EP 354522A), emphysema (U.S. Pat. No. 4499082A), hypertension (EP 315574A) and as factor VII/VIIa inhibitors (WO 8909612A). Kleemann, et. al. (AU A-24693/88) disclose renin-inhibiting 1-amino boronic acid derivatives of formula (3) EQU A.sup.1 --A.sup.2 --HN--CHR.sup.2 --BXR.sup.3 (YR.sup.4) (3)
in which A.sup.1 denotes a radical of formulae (4-8). EQU R.sup.1 NR.sup.6 --CHR.sup.5 --C.dbd.O-- (4) EQU R.sup.1 CHR.sup.12 --CHR.sup.5 --C.dbd.O-- (5) EQU R.sup.1 NR.sup.6 --CHR.sup.5 --CHR.sup.7 --CHR.sup.8 --CHR.sup.9 --C.dbd.O--(6) EQU R.sup.1 CHR.sup.12 --CHR.sup.5 --CHR.sup.7 --CHR.sup.8 --CHR.sup.9 --C.dbd.O-- (7) EQU R.sup.10 --(CH.sub.2).sub.n --CH(CH.sub.2).sub.m R.sup.11 --C.dbd.O--(8)
Despite the foregoing, more efficacious and specific inhibitors of coagulation proteases are needed as potentially valuable therapeutic agents for the treatment of thrombosis. None of the cited references describe or suggest the new thrombin-inhibiting boronic acid derivatives of the present invention.